| 1. |
- Allen, Marie, et al.
(författare)
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Origin of the U87MG glioma cell line : Good news and bad news
- 2016
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Ingår i: Science Translational Medicine. - : American Association for the Advancement of Science (AAAS). - 1946-6234 .- 1946-6242. ; 8:354
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Tidskriftsartikel (refereegranskat)abstract
- Human tumor-derived cell lines are indispensable tools for basic and translational oncology. They have an infinite life span and are easy to handle and scalable, and results can be obtained with high reproducibility. However, a tumor-derived cell line may not be authentic to the tumor of origin. Two major questions emerge: Have the identity of the donor and the actual tumor origin of the cell line been accurately determined? To what extent does the cell line reflect the phenotype of the tumor type of origin? The importance of these questions is greatest in translational research. We have examined these questions using genetic profiling and transcriptome analysis in human glioma cell lines. We find that the DNA profile of the widely used glioma cell line U87MG is different from that of the original cells and that it is likely to be a bona fide human glioblastoma cell line of unknown origin.
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| 2. |
- Birgisson, Helgi, et al.
(författare)
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Microsatellite instability and mutations in BRAF and KRAS are significant predictors of disseminated disease in colon cancer
- 2015
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Ingår i: BMC Cancer. - : Springer Science and Business Media LLC. - 1471-2407 .- 1471-2407. ; 15
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Tidskriftsartikel (refereegranskat)abstract
- Background: Molecular alterations are well studied in colon cancer, however there is still need for an improved understanding of their prognostic impact. This study aims to characterize colon cancer with regard to KRAS, BRAF, and PIK3CA mutations, microsatellite instability (MSI), and average DNA copy number, in connection with tumour dissemination and recurrence in patients with colon cancer. Methods: Disease stage II-IV colon cancer patients (n = 121) were selected. KRAS, BRAF, and PIK3CA mutation status was assessed by pyrosequencing and MSI was determined by analysis of mononucleotide repeat markers. Genome-wide average DNA copy number and allelic imbalance was evaluated by SNP array analysis. Results: Patients with mutated KRAS were more likely to experience disease dissemination (OR 2.75; 95% CI 1.28-6.04), whereas the opposite was observed for patients with BRAF mutation (OR 0.34; 95% 0.14-0.81) or MSI (OR 0.24; 95% 0.09-0.64). Also in the subset of patients with stage II-III disease, both MSI (OR 0.29; 95% 0.10-0.86) and BRAF mutation (OR 0.32; 95% 0.16-0.91) were related to lower risk of distant recurrence. However, average DNA copy number and PIK3CA mutations were not associated with disease dissemination. Conclusions: The present study revealed that tumour dissemination is less likely to occur in colon cancer patients with MSI and BRAF mutation, whereas the presence of a KRAS mutation increases the likelihood of disseminated disease.
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| 3. |
- Bus, Magdalena M., et al.
(författare)
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Forensic Analysis of Mitochondrial and Autosomal Markers Using Pyrosequencing®.
- 2015
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1064-3745 .- 1940-6029. ; 1315, s. 379-396
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Tidskriftsartikel (refereegranskat)abstract
- Forensic casework analyses often face challenges, such as limited genetic material with or without fragmentation and damage. To compensate for low amounts and degradation, shorter amplicons are often applied in the analysis. Also, a change of markers might be necessary using mitochondrial instead of autosomal markers. In addition, forensic research often involves analysis of large number of samples for marker evaluation and population-database compilation. Therefore, a flexible, robust but also rapid method for the detection of variation is highly useful. Pyrosequencing(®) is a rapid, reliable, easy-to-use method for sequence analysis. The method is well suited for rapid forensic analysis of a few targets or analysis of a single target in many samples. It allows sequencing of very short amplicons, which facilitates analysis of degraded DNA. Here we present the use of Pyrosequencing, a robust method for sensitive forensic analysis of mitochondrial DNA, autosomal STRs, and Y-chromosome STRs and SNPs.
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| 4. |
- Coutinho, Alexandra, et al.
(författare)
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Later Stone Age human hair from Vaalkrans Shelter, Cape Floristic Region of South Africa, reveals genetic affinity to Khoe groups
- 2021
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Ingår i: American Journal of Physical Anthropology. - : John Wiley & Sons. - 0002-9483 .- 1096-8644. ; 174:4, s. 701-713
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Tidskriftsartikel (refereegranskat)abstract
- Previous studies show that the indigenous people of the southern Cape of South Africa were dramatically impacted by the arrival of European colonists starting ~400 years ago and their descendants are today mixed with Europeans and Asians. To gain insight on the occupants of the Vaalkrans Shelter located at the southernmost tip of Africa, we investigated the genetic make-up of an individual who lived there about 200 years ago. We further contextualize the genetic ancestry of this individual among prehistoric and current groups. From a hair sample excavated at the shelter, which was indirectly dated to about 200 years old, we sequenced the genome (1.01 times coverage) of a Later Stone Age individual. We analyzed the Vaalkrans genome together with genetic data from 10 ancient (pre-colonial) individuals from southern Africa spanning the last 2000 years. We show that the individual from Vaalkrans was a man who traced ~80% of his ancestry to local southern San hunter–gatherers and ~20% to a mixed East African-Eurasian source. This genetic make-up is similar to modern-day Khoekhoe individuals from the Northern Cape Province (South Africa) and Namibia, but in the southern Cape, the Vaalkrans man's descendants have likely been assimilated into mixed-ancestry “Coloured” groups. The Vaalkrans man's genome reveals that Khoekhoe pastoralist groups/individuals lived in the southern Cape as late as 200 years ago, without mixing with non-African colonists or Bantu-speaking farmers. Our findings are also consistent with the model of a Holocene pastoralist migration, originating in Eastern Africa, shaping the genomic landscape of historic and current southern African populations.
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| 5. |
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| 6. |
- Divne, Anna-Maria, et al.
(författare)
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Forensic analysis of autosomal STR markers using Pyrosequencing
- 2010
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Ingår i: Forensic Science International. - : Elsevier BV. - 1872-4973 .- 1878-0326. ; 4:2, s. 122-129
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Tidskriftsartikel (refereegranskat)abstract
- Short tandem repeats (STRs) are highly variable, and therefore routinely used in forensic investigations for a DNA-based individual identification. The routine assay is commonly performed by size separation using capillary electrophoresis, but alternative technologies can also be used. In this study, a Pyrosequencing assay was developed for analysis of STR markers useful in forensic DNA analysis. The assay was evaluated for 10 different STR loci (CSF1PO, TH01, TPOX, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539 and Penta E) and a total of 114 Swedish individuals were genotyped. This genotyping strategy reveal the actual sequence and variant alleles were seen at several loci, providing additional information compared to fragment size analysis. At the D13S317 locus a T/A SNP located in the last repeat unit was observed in 92% of the genotypes. Moreover, an upstream flanking SNP at locus D7S820, a SNP within the repeats at D3S1358 and D8S1179 and a deletion in the flanking region at locus D5S818 were observed. The Pyrosequencing method was first developed for SNP typing and sequencing of shorter DNA fragments but the method also provides an alternative method for STR analysis of less complex repeats. This assay is suitable for investigation of new markers, a rapid compilation of population data and for confirmation of variant and new alleles.
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| 7. |
- Edlund, Hanna, et al.
(författare)
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DNA extraction and analysis of skeletal remains
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Skeletal remains are often exposed to diverse conditions that affect the biological material in different ways. As a consequence, DNA in aged skeletal remains is often present in minute amounts, highly degraded and contains inhibitors that can affect the amplification reaction. These issues together with the high risk of contamination with exogenous DNA make molecular analysis of old remains a challenging task. Efficient methods for the extraction of DNA that maximise the yield and simultaneously remove inhibitors as well as contaminants would therefore be desirable, both for forensic investigations and for the study of ancient DNA analysis. This study describes the evaluation of three protocols for extraction of DNA from aged skeletal remains. Whole and pulverised samples from a skull and ulna that had been buried for approximately 70 years were treated with bleach and different concentrations of EDTA and proteinase K. The DNA was thereafter extracted using a salting out procedure based on the Wizard® Genomic Purification system. The efficiency of the three protocols was estimated by mtDNA quantification, which revealed that the extracts contained between 0 and 14 110 copies/100mg bone. In general, the ulna bone resulted in higher yields of mtDNA compared to the skull bone. The most efficient extraction protocol was the one involving the highest concentration of EDTA. Soaking the bone samples in commercial bleach prior to extraction was found to be a useful method of eliminating contaminants while retaining sufficient DNA for analysis. Moreover, a maternal relationship was investigated for the remains analysed in this study.
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| 8. |
- Edlund, Hanna, 1977-
(författare)
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Sensitive Identification Tools in Forensic DNA Analysis
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- DNA as forensic evidence is valuable in criminal investigations. Implementation of new, sensitive and fast technologies is an important part of forensic genetic research. This thesis aims to evaluate new sensitive methods to apply in forensic DNA analysis including analysis of old skeletal remains. In Paper I and II, two novel systems for analysis of STRs, based on the Pyrosequencing technology, are presented. In Paper I, Y chromosomal STRs are analysed. Markers on the male specific Y chromosome are especially useful in analysis of DNA mixtures. In Paper II, ten autosomal STRs are genotyped. The systems are based on sequencing of STR loci instead of size determination of STR fragments as in routine analysis. This provides a higher resolution since sequence variants within the repeats can be detected. Determination of alleles is based on a termination recognition base. This is the base in the template strand that is excluded from the dispensation order in the sequencing of the complementary strand and therefore terminates the reaction. Furthermore, skeletal remains are often difficult to analyse, due to damaging effects from the surrounding environment on the DNA and the high risk of exogenous contamination. Analysis of mitochondrial DNA is useful on degraded samples and in Paper III, mtDNA analysis of 700 years old skeletal remains is performed to investigate a maternal relationship. The quantity and quality of DNA are essential in forensic genetics. In Paper IV the efficiency of DNA isolation is investigated. Soaking skeletal remains in bleach is efficient for decontamination but result in a lower DNA yield, especially on pulverised skull samples. In conclusion, this thesis presents novel sequencing systems for accurate and fast analysis of STR loci that can be useful in evaluation of new loci and database assembly as well as the utility of mtDNA in forensic genetics.
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| 9. |
- Edlund, Hanna, et al.
(författare)
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SNP typing using Molecular Inversion Probes
- 2008
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Ingår i: Forensic Science International. - 1875-1768 .- 1875-175X. ; 1:12, s. 473-475
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Tidskriftsartikel (refereegranskat)abstract
- Single nucleotide polymorphism (SNP) typing will be useful in forensic analysis but is limited by the multiplexing capacity of multiple targets in the PCR. A strategy to circumvent this problem can be to use molecular inversion probes (MIPs). MIPs are linear oligonucleotides 70–100 nt in length, with end-segments that are complementary to a target sequence. In this study, 10 SNPs were selected for SNP detection performed by pyrosequencing. The high-multiplexing capacity of MIPs makes this technology very useful for large scale SNP genotyping, but may also be useful for DNA typing in forensic investigations.
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| 10. |
- Edlund, Hanna, et al.
(författare)
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Y chromosomal STR analysis using Pyrosequencing technology
- 2009
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Ingår i: Forensic Science International. - : Elsevier BV. - 1872-4973 .- 1878-0326. ; 3:2, s. 119-124
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Tidskriftsartikel (refereegranskat)abstract
- Analysis of Y chromosome STR markers has proven to be useful in forensic cases where the samples contain a mixture of DNA from several individuals. STR markers are commonly genotyped based on length separation of PCR products. In this study we evaluated if Pyrosequencing can be used as an alternative method for determining Y-STR variants. In total 70 unrelated Swedish males were typed for the Y chromosomal markers (DYS19, DYS389 I-II, DYS390, DYS391, DYS392, DYS393 and DYS438) using Pyrosequencing. Using the 8 markers, 57 unique haplotypes were observed with a discrimination capacity of 0.81. At four loci, the Pyrosequencing analysis revealed sequence variants. The sequence variants were found in the DYS389 II, DYS390, DYS391, and DYS393 loci in frequencies between 1.43% and 14.3%. Pyrosequencing has here been shown to be a useful tool for typing Y chromosomal STRs and the method can provide a complement to conventional forensic Y STR analyses. Moreover, the Pyrosequencing method can be used to rapidly evaluate novel markers.
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